toxicological sciences 135(1), 119–128 2013
doi:10.1093/toxsci/kft142
Advance Access publication June 20, 2013
Metal Allergens Induce Nitric Oxide Production by Mouse Dermal
Fibroblasts via the Hypoxia-Inducible Factor-2α–Dependent Pathway
Toshinobu Kuroishi,*,1 Kanan Bando,*,† Yasuo Endo,* and Shunji Sugawara* *Division of Oral Immunology, Department of Oral Biology and †Division of Orthodontics and Dentofacial Orthopedics, Department of Oral Health and
Developmental Sciences, Tohoku University Graduate School of Dentistry, Sendai 980–8575, Japan
To whom correspondence should be addressed at Division of Oral Immunology, Department of Oral Biology, Tohoku University Graduate School of
Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980–8575, Japan. Fax: +81-22-717-8322. E-mail: [email protected].
1
Received February 28, 2013; accepted June 16, 2013
Nickel (Ni) has been shown to be one of the most frequent metal
allergens. We have already reported a murine metal allergy model
with pathogen-associated molecular patterns (PAMPs) as adjuvants. Interleukin (IL)-1β plays a critical role in our mouse model.
Because nonimmune cells, including fibroblasts, play important
roles in local allergic inflammation, we investigated whether Ni
induces inflammatory responses in mouse dermal fibroblasts
(MDF). We also analyzed the synergistic effects between Ni,
PAMPs, and IL-1β. MDF stimulated with Ni produced a significantly higher amount of nitric oxide (NO) in a dose-dependent
manner. NO production was augmented by costimulation with
IL-1β but not with PAMPs. On the other hand, IL-1β or PAMPs
induced a significantly higher amount of IL-6 production by MDF,
but no augmentation was detected in the presence of Ni. A specific inhibitor for inducible nitric oxide synthase (iNOS) inhibited
Ni-induced NO production. iNOS mRNA expression was significantly higher in MDF stimulated with Ni, IL-1β, or both. A specific
inhibitor for hypoxia-inducible factor (HIF)-2α, but not HIF-1α,
inhibited NO production. Another frequent metal allergen, cobalt,
also induced iNOS expression and NO production by MDF via the
HIF-2α-dependent pathway. The inhibitor for iNOS augmented
ear swelling in Ni allergy mouse model. On the other hand, HIF2α inhibitor attenuates allergic inflammation. These results indicate that metal allergens induce NO production in MDF via the
HIF-2α-dependent pathway and IL-1β augments NO production,
which suggests that the NO induced by metal allergens plays a
pathological role in metal allergies.
Key Words: Ni; IL-1β; HIF; NO; dermal fibroblasts.
Metal allergies have been classified as a type IV allergy
mediated by helper 1 T lymphocytes and generally induce
contact hypersensitivity (CHS) (Thyssen and Menné, 2010).
Nickel (Ni) has been most frequently detected among various metals as a metal allergen (Thyssen and Menné, 2010).
Ni ions and insoluble particles can be taken into cells by ion
channels or active phagocytosis, respectively, and induce many
cellular events (Kasprzak et al., 2003). Ni has been shown to
induce the expression of chemokine receptors and production
of proinflammatory cytokines in human dendritic cells (DCs)
(Antonios et al., 2010; Boislève et al., 2004). It has recently
been reported that human toll-like receptor (TLR) 4, but not the
mouse homologue, is directly activated by Ni and cobalt (Co)
(Raghavan et al., 2012; Schmidt et al., 2010).
Ni exhibits hypoxia-mimicking activity. Ni inhibits prolyl
hydroxylase (PHD), which catalyzes prolyl hydroxylation of
hypoxia-inducible factor (HIF)-α (Ke and Costa, 2006). HIF
is a heterodimeric transcriptional factor consisting of α and β
subunits and binds to hypoxia-responsive element in the promoter region. There are three known active α subunits, HIF-1α,
2α, and 3α, and the single β subunit HIF-1β. Under normoxic
conditions, HIF-α is hydroxylated by PHDs and subsequently
degraded by proteasome. Under hypoxic conditions, PHDs
are inhibited and HIF-α escapes degradation. The stabilized
α subunit translocates to the nucleus and associates with the
β subunit. Ni induces proangiogenic mediators and proinflammatory cytokines via HIF-1α-dependent pathway (Brant and
Fabisiak, 2009).
We previously reported an effective Ni allergy mouse model
that uses lipopolysaccharide (LPS) as an adjuvant (Sato et al.,
2007). In this model, interleukin (IL)-1 is indispensable.
Moreover, LPS augmented the allergy to Ni, not only in the
sensitization but also in the elicitation steps (Kinbara et al.,
2011b). These findings indicated that IL-1 and pathogenassociated molecular patterns (PAMPs) interact with Ni to
elicit both immunological and inflammatory responses.
Nonimmune cells in local tissues, such as fibroblasts, also
play indispensable roles in allergic inflammation. Dermal fibroblasts stimulated by tumor necrosis factor-α and IL-1β have
been shown to activate matrix metalloproteinase-9 production
and the migration of DCs (Saalbach et al., 2010). The activated
dermal fibroblasts produce prostaglandin E2, which induces
Th17-related cytokine IL-23 production from DCs (Schirmer
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et al., 2010). Therefore, it is important to investigate the effects
of metal allergens on dermal fibroblasts in order to clarify the
pathological mechanism of metal allergies.
Nitric oxide (NO) is a multifunctional gas mediator that is
involved in a variety of events (Bogdan, 2001). NO is synthesized by the intracellular enzyme NO synthetase (NOS), which
has three isoforms, neural NOS (nNOS, NOS1) and endothelial NOS (eNOS, NOS3), which are constitutive, and inducible NOS (iNOS, NOS2) (Bogdan, 2001). iNOS is induced in
fibroblasts stimulated by various inflammatory agents including IL-1 and LPS (Jorens et al., 1992; Wang et al., 1996). Ni
was shown to induce iNOS expression in vivo and in vitro
(Cruz et al., 2004; Gupta et al., 2000). HIF also regulates iNOS
expression (Melillo et al., 1995). However, to the best of our
knowledge, no study has shown the direct involvement of HIF
with the induction of iNOS by Ni.
NO shows dual effects in the pathogenesis of CHS (Ross
and Reske-Kunz, 2001). In early phase, concentration of NO
is relatively low, and NO shows proinflammatory effects, such
as tissues damages, vasodilation, and attraction of neutrophils.
Langerhans cells, a DC subtype localized in epithelium, and
keratinocytes express iNOS by stimulation with contact allergens (Ross and Reske-Kunz, 2001). In late phase, activated DC
and macrophages express iNOS and the concentrations of NO
in local inflammatory sites increase. This high NO level shows
anti-inflammatory effects, such as apoptosis of effector cells
and downregulation of the expression of cell adhesion molecules. Because of the dual effects of NO in CHS, contradictory results were reported. NO-releasing cream on normal skin
has been shown to induce significant increases in inflammatory
conditions (Ormerod et al., 1999). NOS inhibitors suppressed
CHS response when the inhibitor was applied at elicitation
phase (Ross et al., 1998). On the other hand, NO-releasing
hydrocortisone shows greater protective effects than its parental compound (Hyun et al., 2004). iNOS downmodulates CHS
response by suppressing DC migration and survival (Sugita
et al., 2010).
In this study, we analyzed NO production by mouse dermal fibroblasts (MDF) stimulated with Ni, PAMPs, and the
proinflammatory cytokine IL-1β. We also analyzed the HIFdependent signals involved in Ni-induced NO production.
Finally, we analyzed in vivo effects of iNOS and HIF inhibitors
on the Ni allergy mouse model.
antibiotics (Antibiotic-Antimycotic, Life Technologies, Carlsbad, CA). The
specimen was cut into 3–5 mm square pieces and placed in a plastic dish (10 cm
diameter), with the dermal side facing the dish. To fix the specimen on the
dish, it was incubated at 37°C for 20 min without medium and then cultured
with 10 ml of culture medium (Dulbecco modified minimum essential medium
containing 10% heat-inactivated fetal bovine serum [FBS] and antibiotics).
Fibroblasts grew after 10–20 days of culture. The culture medium was changed
every 2–3 days, and cells were passaged at 70–80% confluence using 0.25%
Trypsin and 1mM EDTA solution. Cells at passage 5–7 were used for experiments. For in vitro stimulation, cells were seeded in a 24-well culture plate at
2 × 104 per milliliter per well. After 2 days of culture, the culture supernatant
was removed and cells were cultured with the indicated stimuli at 500 µl final
volume. For Western blotting, cells were seeded in a 10-cm-diameter dish at
4 × 105/10 ml/dish. The culture supernatant was removed after 2 days of culture,
and cells were cultured with the indicated stimuli at a final volume of 10 ml.
Cell proliferation and cytotoxicity assay. Cell proliferation was measured with a MTT assay (Cell Counting Kit-8, DOJINDO, Kumamoto, Japan).
For the quantification of cytotoxicity, lactate dehydrogenase (LDH) activity in
the culture supernatant was measured with a cytotoxicity detection kit (Roche
Diagnostic, Indianapolis, IN).
Nitrite assay. Nitrite (NO2−) concentrations were measured using Griess
reagent (1% sulfanilamide, 0.1% n-(1-naphthyl)-ethylenediamine, 2.5% phosphoric acid) with NaNO2 as the standard. The detection limit of the assay was
higher than 2µM nitrite. To analyze significance, ND (not detected) was considered as 2µM.
Measurement of IL-6 in culture supernatants. The amount of IL-6 in the
culture supernatant was measured with a commercial kit (mouse IL-6 ELISA
max standard set, BioLegend).
Quantitative RT-PCR. Cells were lysed in 1 ml Isogen (Nippon Gene,
Toyama, Japan), and total RNA was extracted as described in the instruction
manual. Five hundred nanograms of total RNA was applied to cDNA synthesis
using the Transcriptor First strand cDNA synthesis kit (Roche Diagnostic). Realtime PCR was performed with LightCycler Fast Start DNA Master SYBR Green
I and LightCycler 1.5 Systems (Roche Diagnostic). The primers used for PCR
were as follows: iNOS, forward 5′-GCAAACATCACATTCAGATCCC-3′ and
reverse 5′-TCAGCCTCATGGTAAACACG-3′; glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), forward 5′-CTTTGTCAAGCTCATTTCCTGG-3′
and reverse 5′-TCTTGCTCAGTGTCCTTGC-3′. PCR conditions were 35
cycles at 95°C for 10 s, 60°C for 10 s, and 72°C for 10 s. The product sizes for
iNOS and GAPDH were 150 and 133 bp, respectively. The specificity of the
PCR was confirmed by the product sizes with gel running and melting curve
analysis for each data point. mRNA expression levels were expressed as relative units after normalization by the GAPDH level.
Reagents. HIF-1α and HIF-2α translation inhibitors were purchased from
Merck Millipore (Billerica, MA). LPS from Escherichia coli O55:B5, prepared by Westphal’s method, was purchased from Difco Laboratories (Detroit,
MI). Pam3Cys-Ser-(Lys)4 (Pam3CSK4) was purchased from Merck Millipore.
Recombinant mouse IL-1β was purchased from BioLegend (San Diego, CA).
All other reagents were purchased from Wako pure chemicals (Osaka, Japan),
unless otherwise indicated.
Western blotting. Cells were lysed in RIPA buffer (25mM Tris-HCl,
pH 7.4, 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS)
supplemented with protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO).
After centrifugation, the protein concentrations of cell lysates were determined
by measuring absorbance at 280 nm. Cell lysates were separated by SDSPAGE under reducing conditions. After electrophoresis, gel proteins were
electrophoretically transferred to a polyvinylidene difluoride membrane (BioRad Laboratories, Hercules, CA). The blot was blocked for 1 h with 3% (wt/
vol) skim milk and 0.05% Tween 20 in PBS and incubated with the first Ab
(anti-iNOS, Abcam, Cambridge, UK) followed by the horseradish peroxidase–
conjugated second Ab (Thermo Scientific, Waltham, MA). After washing, the blot
was analyzed with the SuperSignal West Femto Maximum Sensitivity Substrate
(Thermo Scientific) and LAS 4000 mini image analyzer (Fuji Film, Tokyo,
Japan). After stripping with 2M Glycine-HCl (pH 2.8) at room temperature for
1 h, the membrane was incubated again with anti-β-actin (BioLegend) and an
appropriate second Ab to confirm equal loading of the protein.
Cell culture. MDF were isolated from male BALB/cA mice. After shaving the back, skin tissue was removed and rinsed twice with PBS containing
Ni allergy mouse model. Ni allergy mouse model was constructed as
previously reported (Sato et al., 2007). Briefly, mice (female BALB/cA) were
Materials and Methods
NICKEL INDUCES NO PRODUCTION VIA HIF-2α
121
sensitized with an ip injection of 250 µl of solution containing 0.5mM NiCl2
and 0.5 µg/ml LPS. Ten days after sensitization, the mice were challenged with
20 µl of 1mM NiCl2 by an intradermal injection into the both pinnas. Ear swelling was measured at the indicated times using Peacock dial thickness gouge.
Statistical analysis. Experiment values were given as the mean ± SD of a
single experiment performed in triplicate. All of the experiments in this study
were performed at least thrice to confirm the reproducibility of the results.
The data shown are representative results. Statistical analysis was performed
with the unpaired t-test between two groups, or a one-way ANOVA using
Bonferroni’s (to compare all groups) or Dunnet’s (to compare between the
control and others) method between three or more groups, and p < 0.05 was
considered significant.
Results
Cytotoxic Effects of Ni2+ on MDF
At first, we analyzed the cytotoxic effects of NiCl2 (Ni2+) on
MDF. As shown in Figure 1A, MDF proliferation was inhibited
in a Ni2+ dose-dependent manner. In the presence of 100µM
Ni2+, MDF proliferation after 4 days of culture was approximately 65% of the medium (0µM of Ni2+). To confirm that the
lower cell proliferation is cytotoxic but not cytostatic effects,
we also measured LDH activity in the culture supernatants as
an indicator of cytoplasmic leakage. Because FBS shows high
LDH activity, we used culture medium with 1% FBS to reduce
background levels in the measurements of LDH activity. In the
presence of 50 and 100µM Ni2+, approximately 40% cytotoxicity was measured after 4 days of culture (Fig. 1B).
These results indicated that a higher concentration of Ni2+
had cytotoxic effects on MDF.
NO Production Induced By Ni2+ and Augmentation By IL-1β
Next, we measured nitrite concentrations in the culture
supernatants as an indicator of NO production. As shown in
Figure 2A, nitrite concentrations in the culture supernatants of
MDF stimulated with Ni2+ for 4 days increased in a Ni2+ dosedependent manner. The concentration of nitrite with 100µM
Ni2+ was approximately 20µM, which was significantly higher
than that of the control (0µM of Ni2+). A significant amount
of nitrite was detected after 3 days of culture and increased
in a time-dependent manner up to 4 days of culture (Fig. 2B).
Moreover, another Ni salt, NiSO4, but not another chloride,
CaCl2, also induced NO production by MDF (data not shown).
To exclude the possibility of cytoplasmic leakage, we measured
nitrite concentrations in whole cell lysates. However, no nitrite
was detected even in whole cell lysates prepared from MDF
stimulated with IL-1β (data not shown). We also measured
NO production in MDF stimulated with other chemical irritants, such as dimethyl sulfoxide, HCl, NaOH, SDS, and Triton
X-100. However, no nitrite was detected in culture supernatants
of MDF stimulated with chemical irritants (data not shown).
We previously reported that PAMPs were effective adjuvants
for a Ni allergy and that IL-1 played an important role in the
pathogenesis of a Ni allergy (Sato et al., 2007). We then analyzed
Fig. 1. Cytotoxic effects of Ni2+ on MDF. Cells were cultured in medium
containing 10% (A) or 1% (B) FBS with the indicated amount of NiCl2 at
a final volume of 500 µl for 4 days. (A) Cell proliferation was measured by
a MTT assay. The results were expressed as % cell proliferation after being
normalized to the values with 0µM NiCl2. (B) Cytotoxicity was determined by
measuring LDH activity in culture supernatants. The results were expressed as
% cytotoxicity after being normalized to the values with 0µM NiCl2 as 0% and
those with 2% Triton X-100 as 100%. The results were expressed as the mean
± SD of a single experiment performed in triplicate. Means without a common
symbol differ, p < 0.05.
the synergistic effects of PAMPs, such as LPS (a TLR4 ligand),
Pam3CSK4 (a TLR2 and 1 ligand), and IL-1β to Ni2+-induced
NO production. As shown in Figure 3A, only a traceable amount
of nitrite was detected in the culture supernatant of MDF
stimulated with IL-1β alone at 10 and 100 ng/ml. In the case of
costimulation with Ni2+ and IL-1β, nitrite concentrations were
significantly higher than those with Ni2+ alone. On the other
hand, LPS and Pam3CSK4 alone did not induce NO production,
and only a slight augmentation was detected when MDF
were costimulated with higher concentrations (100 ng/ml) of
LPS or Pam3CSK4 and Ni2+ (Fig. 3A). We also analyzed IL-6
production. Figure 3B shows that LPS, Pam3CSK4, and IL-1β
alone induced IL-6 production in MDF. Higher concentrations
(100 ng/ml) of LPS and Pam3CSK4 induced approximately
60 ng/ml of IL-6. On the other hand, IL-1β induced a higher
amount of IL-6 (approximately 130 ng/ml) even at a lower
concentration (10 ng/ml). Regardless of stimulation, IL-6
production decreased significantly in the presence of Ni2+,
which may have been caused by the cytotoxic effects of Ni2+.
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IL-1β (Fig. 4D). No iNOS protein was detected in MDF with
medium and IL-1β.
These results indicated that iNOS was induced by Ni2+ in
MDF.
HIF-2α, But Not HIF-1α, Mediates iNOS Induction and NO
Production
Because HIF mediates various cellular events induced by Ni
(Ke and Costa, 2006), and iNOS expression is regulated by HIF
(Melillo et al., 1995), we analyzed the effects of HIF-α inhibitors. As shown in Figures 5A and B, nitrite concentrations were
significantly decreased in the presence of the HIF-2α inhibitor,
but not the HIF-1α inhibitor, regardless of stimulation with Ni2+
alone (Fig. 5A) or with Ni2+ and IL-1β (Fig. 5B). Moreover,
the HIF-2α inhibitor significantly decreased the expression levels of iNOS mRNA induced by Ni2+ alone or Ni2+ and IL-1β
(Fig. 5C). iNOS protein was also decreased in the presence of
HIF-2α inhibitor (Fig. 5D). No additive effects of HIF-1α and
HIF-2α inhibitors were detected (data not shown). The expression of iNOS mRNA was dependent on HIF-2α even with
shorter period (8 h, data not shown).
These results indicated that HIF-2α, but not HIF-1α, was
involved with iNOS induction and NO production by Ni2+stimulated MDF.
Fig. 2. NO production by MDF stimulated with Ni2+. Cells were cultured
in medium containing 10% FBS with the indicated amount of NiCl2 at a final
volume of 500 µl. Nitrite concentrations were measured with Griess reagent.
(A) Cells were stimulated for 4 days. ND, not detected. Means without a common symbol differ, p < 0.05. (B) Cells were stimulated with 100µM of NiCl2
for the indicated period. *p < 0.05, significantly different from the medium.
The results were expressed as the mean ± SD of a single experiment performed
in triplicate.
These results indicated that Ni2+ induced NO production in
MDF in a dose- and time-dependent manner and IL-1β augmented it.
iNOS Induction By Ni2+
To analyze whether iNOS was involved in the NO production induced by Ni2+, we measured nitrite concentrations in
the presence or absence of the NOS inhibitors, NG-nitro-Larginine methyl ester (L-NAME, eNOS inhibitor) and L-N6(1-iminoethyl)lysine (L-NIL, iNOS inhibitor). As shown in
Figures 4A and B, L-NIL, but not L-NAME, significantly inhibited NO production by MDF stimulated with Ni2+ (Fig. 4A)
or Ni2+ and IL-1β (Fig. 4B). We also measured iNOS mRNA
expression levels by quantitative RT-PCR (Fig. 4C). IL-1β
alone induced traceable amounts of iNOS mRNA expression.
Ni2+ induced significantly higher expression levels of iNOS
mRNA, and costimulation with Ni2+ and IL-1β greatly induced
the expression of iNOS mRNA. Moreover, iNOS protein was
detected in MDF stimulated with Ni2+, and a higher amount
of iNOS protein was detected by costimulation with Ni2+ and
NO Production Induced By Co2+
We analyzed other metal cations that have been reported as
metal allergens. We tested Co2+, Cr2+, and Cu2+. Palladium (Pd)
has also been reported as a metal allergen, and we previously
showed that Ni and Pd exhibited cross reactivity (Kinbara et al.,
2011a). However, because of its low solubility in neutral pH,
we could not test Pd2+. No nitrite was detected in the culture
supernatant of MDF stimulated with Cr2+ or Cu2+ (data not
shown). As shown in Figure 6A, Co2+ had cytotoxic effects on
MDF. In the presence of 100µM of Co2+, MDF proliferation
after 4 days of culture was approximately 32% of the medium
(0µM of Co2+). Even at a lower concentration (12.5µM), cell
proliferation was approximately 60%. Nitrite concentrations
increased in a Co2+ dose-dependent manner up to 50µM and
significantly decreased with 100µM of Co2+ (Fig. 6B). When
cells were costimulated with Co2+ and IL-1β, NO production
was significantly augmented. Nitrite concentrations with 100µM
of Co2+ and IL-1β were as same as those with 50µM of Co2+ and
IL-1β. The inhibitor of HIF-2α, but not HIF-1α, significantly
decreased NO production and the expression of iNOS mRNA
induced by Co2+ alone or Co2+ and IL-1β (Figs. 6C and D).
These results indicated that Co2+ also induced iNOS induction
and NO production by MDF via the HIF-2α-dependent pathway.
iNOS Inhibitor Augments, But HIF-2α Inhibitor Attenuates
Allergic Inflammation in Ni Allergy Mouse Model
Finally, we analyzed the in vivo effects of iNOS and HIF-α
inhibitors on Ni allergy mouse model. Each inhibitor was
NICKEL INDUCES NO PRODUCTION VIA HIF-2α
123
Fig. 3. IL-1β augments NO production induced by Ni2+. Cells were cultured in medium containing 10% FBS with the indicated amount of NiCl2 at a final
volume of 500 µl for 4 (A) or 1 (B) day. (A) Nitrite concentrations in the culture supernatants were measured with Griess reagent. (B) IL-6 concentrations in the
culture supernatants were measured by ELISA. The results were expressed as the mean ± SD of a single experiment performed in triplicate. ND, not detected.
*p < 0.05, **p < 0.01, significantly different from no stimulation (0 ng/ml). #p < 0.05, significantly different from 0µM of NiCl2.
coadministrated with Ni at elicitation step. As shown in
Figure 7, ear swelling was significantly increased in the presence of 100µM of iNOS inhibitor, L-NIL. Surprisingly, HIF-2α
inhibitor, but not HIF-1α inhibitor, significantly inhibited the
ear swelling.
These results indicated that iNOS and HIF-2α showed antiand proinflammatory effects on the Ni allergy mouse model,
respectively.
Discussion
We showed that iNOS expression and NO production were
induced by Ni2+ in MDF via the HIF-2α-dependent pathway.
Several studies have reported Ni-induced NO production in
vivo and in vitro (Cruz et al., 2004; Gupta et al., 2000). Ni is a
well-known hypoxia-mimicking agent that activates HIF pathways (Ke and Costa, 2006). iNOS is a typical HIF target gene
(Melillo et al., 1995). Ni oxide nanoparticles, as well as Ni2+,
induced HIF-1α accumulation and its nuclear translocation
in the human lung epithelial cell line NCI-H460 (Pietruska
et al., 2011). Primary cells, such as human endothelial cells
(HUVEC) and human lung fibroblasts, also showed the activation of HIF pathways by stimulation with Ni2+ (Brant and
Fabisiak, 2009; Viemann et al., 2007). These findings support our present results that Ni2+ induced iNOS expression
in MDF via a HIF-dependent mechanism. To the best of our
knowledge, this is the first study to show the Ni-HIF-iNOS
axis directly.
Although HIF-1α and HIF-2α have overlapping functions,
many studies have reported unique and sometimes opposing
roles for both HIF-α subunits. Takeda et al. (2010) reported
the opposing roles of HIF-1α and 2α in NO production by
mouse peritoneal macrophages under hypoxia conditions in
which HIF-1α induced iNOS expression, but HIF-2α regulated
arginase 1 expression, which catabolizes L-arginine and
competes with iNOS for their common substrate. Although the
cell type was different, their report conflicts with our present
results that HIF-2α, but not HIF-1α, regulated iNOS expression
in MDF. Several studies have reported differential stabilities
between HIF-1α and 2α. HIF-1α is transiently stabilized under
low hypoxia (1% O2), whereas HIF-2α is stabilized under
moderate hypoxia (5% O2) during prolong periods (HolmquistMengelbier et al., 2006). In this study, iNOS expression
induced by Ni2+ was dependent on HIF-2α in not only longer
(3 days) but also shorter (8 h) periods of time. The difference
between “actual hypoxia” and “a hypoxia-mimicking agent”
may affect the preference of HIF-α. Moreover, the different
regulatory mechanisms of HIF-α have also been reported.
The efficiency of HIF-α hydroxylation by PHDs and HIF
asparaginyl hydroxylase was shown to differ between HIF-1α
and HIF-2α (Appelhoff et al., 2004; Koivunen et al., 2004).
Koh et al. (2011) reported that hypoxia-associated factor
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Fig. 4. iNOS is involved with NO production stimulated with Ni2+ and IL-1β. Cells were cultured in medium containing 10% FBS with the indicated amount
of stimuli. (A and B) Cells were stimulated with 100µM NiCl2 and the NOS inhibitors, L-NIL or L-NAME, in the presence (B) or absence (A) of 10 ng/ml of
IL-1β for 4 days. Nitrite concentrations were measured with Griess reagent. (C) Cells were stimulated with 100µM of NiCl2, 10 ng/ml of IL-1β, or both for 3 days.
iNOS mRNA expression levels were determined by quantitative RT-PCR. The results were expressed as relative units after normalization by the GAPDH level.
The results were expressed as the mean ± SD of a single experiment performed in triplicate. *p < 0.05, **p < 0.01, significantly different from the medium. (D)
Cells were stimulated with 100µM of NiCl2, 10 ng/ml of IL-1β, or both in a final volume of 10 ml for 4 days. The whole cell lysate (50 µg protein/well) was applied
to Western blotting.
mediated HIF-1α degradation but not HIF-2α activation. It is
important to further investigate the stabilization and activation
mechanisms of HIF-α induced by Ni.
IL-1β significantly augmented NO production by MDF
stimulated with Ni2+. On the other hand, only slight augmentations were induced by the TLR ligands, LPS and Pam3CSK4.
IL-6 production was also higher in MDF stimulated with IL-1β
than with the TLR ligands. IL-1R and TLR share many signal
transduction molecules, such as NF-κB and mitogen-activated
protein kinase (MAPK) (O’Neill and Bowie, 2007), which suggests that signal strength, but not signal type, contributes to this
augmentation. We previously reported that LPS reduces the
minimum concentration of Ni to induce allergic inflammation
in the elicitation step (Kinbara et al., 2011b). LPS is a wellknown inducer of IL-1β (Alheim and Bartfai, 1998), indicating
that IL-1β induced by LPS cooperates with Ni in the elicitation step. The interaction between the HIF, MAPK, and NF-κB
pathways has been reported, suggesting that the HIF signal
induced by Ni and MAPK and/or the NF-kB signal induced by
IL-1β have synergistic/additive effects.
iNOS inhibitor, L-NIL, augmented significantly ear
swelling in Ni allergy mouse model, indicating that NO has
anti-inflammatory effects in our mouse model. This result agrees
with other studies, which reported anti-inflammatory effects of
NO. Sugita et al. (2010) reported that CHS response to hapten
increased by the application of L-NIL, and iNOS expressed in
cutaneous DC suppressed the migration and survival of DCs.
NO-releasing hydrocortisone shows faster and greater protective
effects than its parental compound (Hyun et al., 2004). The
cell adhesion molecules were downregulated by NO donor in
HUVEC and smooth muscle cells (Shin et al., 1996). Although
NO is highly diffusible, the biological effects of NO are not
restricted to the site of its production. NO metabolites, such
as the low molecular weighted S-nitrosothiols, S-nitrosylated
proteins, and nitrosyl-metal complexes, can act as long-distance
NO vehicles (Bogdan, 2001). These observations suggest that
NO derived from Ni-stimulated dermal fibroblasts shows antiinflammatory effects in Ni allergy.
On the other hand, HIF-2α inhibitor reduced significantly
ear swelling in Ni allergy mouse model. This surprising result
is not keeping with the results that Ni-stimulated MDF produces NO via HIF-2α-dependent manner, and the inhibitor
for iNOS augments ear swelling in Ni allergy mouse model.
It was reported that mice lacking HIF-2α in myeloid cells are
NICKEL INDUCES NO PRODUCTION VIA HIF-2α
125
Fig. 5. HIF-2α is involved with NO production stimulated with Ni2+ and IL-1β. Cells were cultured in medium containing 10% FBS with the indicated
amount of stimuli. (A and B) Cells were stimulated with 100µM NiCl2 and the indicated amount of HIF-1α or 2α inhibitors in the presence (B) or absence (A) of
10 ng/ml of IL-1β for 4 days. Dimethyl sulfoxide (DMSO) was used as the vehicle control. Nitrite concentrations were measured with Griess reagent. (C) Cells
were stimulated with 100µM NiCl2 and 5µM of HIF-1α or 2α inhibitors in the presence or absence of 10 ng/ml of IL-1β for 3 days. A total of 0.5% of DMSO
was used as the vehicle control. iNOS mRNA expression levels were determined by quantitative RT-PCR. The results were expressed as relative units after normalization by the GAPDH level. The results were expressed as the mean ± SD of a single experiment performed in triplicate. *p < 0.05, **p < 0.01, significantly
different from the medium. (D) Cells were stimulated with 100μM of NiCl2 and 10 ng/ml of IL-1β with 5μM of HIF-1α or 2α inhibitors in a final volume of 10 ml
for 4 days. DMSO was used as the vehicle control. The whole cell lysate (50 μg protein/well) was applied to Western blotting.
resistant to endotoxin shock and display a significantly lower
inflammatory responses to cutaneous and peritoneal irritants
(Imtiyaz et al., 2010). Bone marrow–derived macrophages
from the conditional knockout mice produce significantly
lower amount of proinflammatory cytokines, such as IL-1β,
IL-6, and IL-12, indicating that that HIF-2α regulates proinflammatory cytokines expression. These observations suggest
that, even though the HIF-2α inhibitor downregulates the NO
production by Ni-stimulated MDF, effects of lower amount of
NO are masked by effects of lower amount of proinflammatory
cytokines. Further studies will be needed to elucidate the precious roles of HIF-2α in the pathogenesis of Ni allergy.
IL-6 production decreased significantly in the presence of
Ni even though costimulation with TLR ligands or IL-1β.
Although we did not rule out the precious mechanisms, it
may have been caused by the cytotoxic effects of Ni. On the
other hand, Gao et al. (2010) reported that Ni induces IL-6
production by human lung fibroblasts. They also showed that
IL-6 production increased significantly by costimulation with
Ni and TLR2 ligand. This discrepancy is possibly caused
by experimental system. They used human lung fibroblasts
with serum-free medium. It is well known that serum affects
cytokine production. Further studies are needed to verify the
effects of Ni on the IL-6 production.
Dermal fibroblasts regulate DCs function by producing
inflammatory cytokines and prostanoids (Saalbach et al., 2010;
Schirmer et al., 2010), indicating that dermal fibroblasts are
one of the important players in the pathogenesis of skin inflammation. Because Ni is challenged intradermally to ear pinnas,
it can directly stimulate the dermal fibroblasts in our mouse
model (Sato et al., 2007). In vitro study with human full-thickness skin shows that a small but considerable amount of Ni
is able to penetrate the stratum corneum and reach at dermis
(Fullerton et al., 1988). Moreover, volunteers cutaneously
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KUROISHI ET AL.
Fig. 6. NO production by MDF stimulated with Co2+. Cells were cultured in medium containing 10% FBS with the indicated amount of stimuli. (A) Cells
were stimulated with the indicated amount of CoCl2 for 4 days. Cell proliferation was measured by a MTT assay. The results were expressed as % cell proliferation after being normalized to the values with 0μM CoCl2. Means without a common symbol differ, p < 0.05. (B) Cells were stimulated with the indicated amount
of CoCl2 in the presence or absence of 10 ng/ml of IL-1β for 4 days. Nitrite concentrations were measured with Griess reagent. The results were expressed as
the mean ± SD of a single experiment performed in triplicate. ND, not detected. Means without a common symbol differ between each group. (C) Cells were
stimulated with 50μM CoCl2 and 5μM of HIF-1α or 2α inhibitors in the presence or absence of 10 ng/ml of IL-1β for 4 days. A total of 0.5% of DMSO was used
as the vehicle control. Nitrite concentrations were measured with Griess reagent. The results were expressed as the mean ± SD of a single experiment performed
in triplicate. *p < 0.05, **p < 0.01 significantly different from the medium. (D) Cells were stimulated with 50μM CoCl2 and 5μM of HIF-1α or 2α inhibitors in
the presence or absence of 10 ng/ml of IL-1β for 3 days. A total of 0.5% of DMSO was used as vehicle control. iNOS mRNA expression levels were determined
by quantitative RT-PCR. The results were expressed as relative units after normalization by the GAPDH level. The results were expressed as the mean ± SD of a
single experiment performed in triplicate. *p < 0.05, significantly different from DMSO.
exposed to Co were shown to have higher urinary Co concentrations (Scansetti et al., 1994). These observations indicate
that Ni and Co can directly stimulate dermal tissues and support the involvement of dermal fibroblasts in the pathogenesis
of metal allergies. On the other hand, as a front line of biological defense, epidermal cells, such as Langerhans cells and
keratinocytes, are likely to be the first responder to Ni (Ross and
Reske-Kunz, 2001). These cells express iNOS mRNA at elicitation step of CHS induced by contact allergen 2,4-dinitrofluorobenzene (Ross et al., 1998). Ni also induces the expression
of iNOS in skin-derived DC (Cruz et al., 2004). Although Ni
induces inflammatory cytokines and cell adhesion molecules in
human and mouse keratinocytes (Corsini et al., 1998; Guéniche
et al., 1994), it has not been clarified whether Ni induces iNOS
expression and NO production in the keratinocytes. Further
investigations are needed to elucidate the interaction between
Ni and skin tissues including epidermis and dermis.
Because we have the Ni allergy mouse model and it is
relatively easy to progress to in vivo experiments, we used
MDF in this study. On the other hand, it is important to evaluate
whether human dermal fibroblasts respond to Ni same as MDF.
The relevance of our data obtained in MDF for the pathogenesis
of human Ni allergy needs to be validated.
The only effective method to prevent metal allergies is to
reduce or avoid metal exposure (Thyssen and Menné, 2010).
However, it is not easy to identify and avoid all metal allergens in various daily items. In this study, we clearly showed
that two major metal allergens, Ni and Co, induced NO production by dermal fibroblasts. Although the mechanisms
are still unclear, the modulation of this multifunctional gas
mediator may help prevent a metal allergy. Further investigations are needed to elucidate the crucial roles of NO derived
from metal-stimulated dermal fibroblasts in the pathogenesis
of metal allergies.
NICKEL INDUCES NO PRODUCTION VIA HIF-2α
127
Fig. 7. Effects of iNOS and HIF-α inhibitors on the allergic inflammation in Ni allergy mouse model. Mice were sensitized with an ip injection
of 250 μl of solution containing 0.5mM NiCl2 and 0.5 μg/ml LPS. Ten days
after sensitization, ear pinnas were challenged with an intradermal injection
of 20 μl of 1mM NiCl2 with 100μM of L-NIL and 5μM of HIF-1α or 2α
inhibitor. A total of 0.5% of DMSO was used as vehicle control to HIF-α
inhibitors. Ear swelling was measured at 48 h after challenge. The results
were expressed as the mean ± SD of four pinnas. Means without a common
symbol differ, p < 0.01.
Funding
Japan Society for the Promotion of Science KAKENHI
(23792140 to T.K., 24659835 to Y.E., 24390407 to S.S.); The
Nakatomi Foundation (to T.K.).
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